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  • Original Paper
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Phosphorylation and ubiquitination of oncogenic mutants of β-catenin containing substitutions at Asp32

Oncogene volume 23, pages 4839–4846 (2004)Cite this article

Abstract

β-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3β (GSK-3β)-dependent phosphorylation of Ser/Thr residues in the N-terminus of the protein, followed by ubiquitination and proteosomal degradation. In human and rodent cancers, mutations that substitute one of the critical Ser/Thr residues in the GSK-3β region of β-catenin stabilize the protein and activate β-catenin/TCF/LEF target genes. This study examined three oncogenic β-catenin mutants from rat colon tumors containing substitutions adjacent to amino-acid residue Ser33, a key target for phosphorylation by GSK-3β. Compared with wild-type β-catenin (WT), the β-catenin mutants D32G, D32N, and D32Y strongly activated TCF-4-dependent transcription in HEK293 cells, and there was accumulation of β-catenin in the cell lysates. Immunoblotting with phosphospecific antibodies indicated that there was little if any effect on the phosphorylation of Ser37, Thr41 or Ser45; however, the phosphorylation of Ser33 appeared to be affected in the β-catenin mutants. Specifically, antiphospho-β-catenin 33/37/41 antibody identified high, intermediate and low expression levels of phosphorylated β-catenin in cells transfected with D32G, D32N and D32Y, respectively. Experiments with the proteosome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) revealed ubiquitinated bands on all three mutant β-catenins, as well as on WT β-catenin. The relative order of ubiquitination was WT>D32G>D32N>D32Y, in parallel with findings from the phosphorylation studies. These results are discussed in the context of previous studies, which indicated that amino-acid residue D32 lies within the ubiquitination recognition motif of β-catenin.

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References

  • Aberle H, Bauer A, Stappert J, Kispert A and Kemler R . (1997). EMBO J, 16, 3797–3804.

  • Blum CA, Xu M, Orner GA, Fong AT, Bailey GS, Stoner GD, Horio DT and Dashwood RH . (2001). Carcinogenesis, 22, 315–320.

  • Blum CA, Tanaka T, Zhong X, Li Q, Dashwood W-M, Pereira C, Xu M and Dashwood RH . (2003). Mol. Carcinog., 36, 195–203.

  • Clements WM, Wang J, Sarnaik A, Kim OJ, MacDonald J, Fenoglio-Preiser C, Groden J and Lowy AM . (2002). Cancer Res., 62, 3503–3506.

  • Conacci-Sorrell M, Zhurinsky J and Ben-Ze'ev A . (2002). J. Clin. Invest., 109, 987–991.

  • Dashwood RH, Suzui M, Nakagama H, Sugimura T and Nagao M . (1998). Cancer Res., 58, 1127–1129.

  • Dashwood W-M, Orner GA and Dashwood RH . (2002). Biochem. Biophys. Res. Commun., 296, 584–588.

  • Espinosa L, Ingles-Esteve J, Aguilera C and Bigas A . (2003). J Biol. Chem., 278, 32227–32235.

  • Fujita Y, Krause G, Scheffner M, Zechner D, Leddy HE, Behrens J, Sommer T and Birchmeier W . (2002). Nature Cell Biol., 4, 222–231.

  • Hagen T and Vidal-Puig A . (2002). Biochem. Biophys. Res. Commun., 294, 324–328.

  • Hajra KM and Fearon ER . (2002). Genes Chromosomes Cancer, 34, 255–268.

  • Hirohashi S . (1998). Am. J. Pathol., 153, 333–339.

  • Huelsken J and Behrens J . (2002). J. Cell. Sci., 115, 3977–3978.

  • Ikeda S, Kishida S, Yamamoto H, Murai H, Koyama S and Kikuchi A . (1998). The EMBO J., 17, 1371–1384.

  • Ilyas M, Tomlinson IP, Rowan A, Pignatelli M and Bodmer WF . (1997). Proc. Natl. Acad. Sci. USA, 94, 10330–10334.

  • Kinzler KW and Vogelstein B . (1997). Nature, 386, 761–763.

  • Li Q, Dixon BM, Al-Fageeh M, Blum CA and Dashwood RH . (2002). Gene, 283, 255–262.

  • Li, Q, Dashwood W-M, Zhong X, Al-Fageeh and Dashwood RH . (2004). Genomics, 83, 231–242.

  • Liu C, Kato Y, Zhang Z, Do VM, Yankner BA and He X . (1999). Proc. Natl. Acad. Sci. USA, 96, 6273–6278.

  • Liu J, Stevens J, Rote CA, Yost HJ, Hu Y, Neufeld KL, White RL and Matsunami N . (2001). Mol. Cell, 7, 927–936.

  • Lustig B, Jerchow B, Sachs M, Weiler S, Pietsch T, Karsten U, van de Wetering M, Clevers H, Schlag PM, Birchmeier W and Behrens J . (2002). Mol. Cell. Biol., 22, 1184–1193.

  • Matsuzawa S and Reed JC . (2001). Mol. Cell, 7, 915–926.

  • Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B and Kinzler KW . (1997). Science, 275, 1787–1790.

  • Nusse R . (2003). http://www.stanford.edu/~rnusse/arm/bcatmut.html.

  • Oving IM and Clevers HC . (2002). Eur. J. Clin. Invest., 32, 448–457.

  • Polakis P . (2000). Genes Dev., 14, 1837–1851.

  • Porfiri E, Rubinfeld B, Albert I, Hovanes K, Waterman M and Polakis P . (1997). Oncogene, 15, 2833–2839.

  • Rubinfeld B, Robbins P, El-Gamil M, Albert I, Porfiri P and Polakis P . (1997). Science, 275, 1790–1792.

  • Sparks AB, Morin PJ, Vogelstein B and Kinzler KW . (1998). Cancer Res., 58, 1130–1134.

  • Suzui M, Ushijima T, Dashwood RH, Yoshimi N, Sugimura T, Mori H and Nagao M . (1999). Mol. Carcinog., 24, 232–237.

  • Takahashi M, Fukuda K, Sugimura T and Wakabayashi K . (1998). Cancer Res., 58, 42–46.

  • Van Gassen G, De Jonghe C, Nishimura M, Yu G, Kuhn S, St George-Hyslop P and Van Broeckhoven C . (2000). Mol. Med., 6, 570–580.

  • Wang Z, Vogelstein B and Kinzler KW . (2003). Cancer Res., 63, 5234–5235.

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Acknowledgements

Expression constructs for WT β-catenin, TCF-4, TOPflash and FOPflash as well as α-ABC antibody were kindly provided by Dr Marc van de Wetering and Dr Hans Clevers, University Hospital Utrecht, The Netherlands. Dr Akira Kikuchi of Hiroshima University School of Medicine generously supplied a construct for rAxin. We thank Dr Robert Tanguay for use of the gel scanner and associated software. We are grateful for services provided by the Center for Gene Research and Biotechnology, Oregon State University. This work was supported in part by NIH Grants CA65525, CA80176 and CA90890.

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  1. Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, 97331-6512, OR, USA

    Mohamed Al-Fageeh, Qingjie Li, W Mohaiza Dashwood, Melinda C Myzak & Roderick H Dashwood

  2. Department of Environmental & Molecular Toxicology, Oregon State University, Corvallis, 97331-6512, OR, USA

    Roderick H Dashwood

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  1. Mohamed Al-Fageeh
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Correspondence to Roderick H Dashwood.

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Al-Fageeh, M., Li, Q., Mohaiza Dashwood, W. et al. Phosphorylation and ubiquitination of oncogenic mutants of β-catenin containing substitutions at Asp32. Oncogene 23, 4839–4846 (2004). https://doi.org/10.1038/sj.onc.1207634

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